The goal of this project is to define the interactions between RNA polymerase II, the basal transcription factors, and the chromatin template that lead to accurate transcription initiation and productive elongation. Using the yeast Saccharomyces cerevisiae as a model, several fundamental aspects of gene expression will be studied. This project period will focus on the mechanisms for targeting co-transcriptional histone methylation of histone H3K4 and H3K36. It is clear that the Bur1 kinase promotes transcription through chromatin, as the requirement for Bur1 can be bypassed by mutating H3K36, or by deleting the H3K36 methyltransferase Set2 or several other chromatin-related factors. The first specific aim is to further probe the substrates and functions of Bur1. Experiments in the second aim will explore the interactions between the methylations at H3K4 and H3K36. Although these modifications have typically been considered as separate events, preliminary data indicates they are not independent. It is still not clear exactly how these modifications affect transcription, so their role in affecting transcription elongation will be probed by a series of genetic and molecular experiments. H3K4 tri-methylation is localized near promoters and it has been proposed that the Set1/COMPASS complex is recruited to the Serine 5 phosphorylated form of the RNA polymerase II C-terminal domain (CTD). However, there is no experimental evidence for a direct interaction between COMPASS and the CTD. The third specific aim will be to test whether COMPASS can directly bind specific phosphorylated forms of the CTD, and if so, which subunits are responsible. The fourth specific aim will follow up on preliminary data suggesting that the Rpb4 subunit of RNA polymerase II is involved in mediating interactions between the transcription complex and the elongation factor Spt6. Protein interaction experiments will test for direct interactions between Spt6 and the Rpb4/7 heterodimer, while chromatin immunoprecipitation experiments will explore the effect of Rpb4 deletion on histone deposition and modification. Affinity chromatography and mass spectrometry will be used to identify other Rpb4/7 binding proteins to see if other chromatin-related complexes interact with RNA polymerase II through this subcomplex. In the last specific aim, initiation and elongation complexes formed under various conditions will be purified on immobilized templates and then analyzed by mass spectometry. Although it is possible some new factors associated with transcription complexes will be identified, what is of greater interest is the exchange of factors that are likely to occur at various stages of transcription. The experiments in these five specific aims will significantly increase our understanding of the RNA polymerase II transcription reaction and its interactions with the chromatin template. This fundamental knowledge is essential for understanding how mutations in transcription factors and histone modifying enzymes lead to diseases such as cancer and developmental defects. [unreadable] [unreadable] [unreadable]